Thioredoxin peroxidase(TPx) is a new type of peroxidase that belongs to peroxiredoxin family. Tpx contains two essential cysteine residues that directly participate in catalytic cycle. Tpx exists as a head-tail dimer and its catalytic function requires cysteine residues from both subunits. To study cellular function of Tpx, we prepared a dominant negative mutant form of Tpx by mutating the two conserved cysteine residues to serine residues. As expected, the mutant polypeptide formed a dimer with wild type, incapacitating the activity of wild type. Overexpression of wild-type mutant TPx in NIH3T3 cells partially reduced the transient increase of H2O2 generated in response to TNFalpha, whereas overexpression of mutant TPx potentiated the H2O2 response, indicating that TPx is directly involved in regulation of the intracellular level of H2O2. We then investigated the effect of their expression on TNFalpha-induced activation of NF-kB and MAP kinases and apoptosis of HeLa cells. Overexpression of wild type resulted in a suppression of the phosphorylation of IkappaB and subsequently the transcriptional activity of NFkappaB, whereas mutant expression had opposite effects. The apoptosis, triggered by TNFalpha and cyclohexamide treatment, was dramatically accelerated in cells expressing the mutant, whereas it was inhibited by wild-type enzyme. Furthermore, TPx modulated the activation of FLICE, the most upstream caspase in TNFalpha-induced apoptosis. Expression of wild-type Tpx did not affect ERK activity, whereas dominant mutant expression inhibited ERK activation. These results suggest that TPx may function as a physiological modulator of second messenger H2O2 produced in response to TNFalpah and that different signaling cascades exhibit different sensitivity to intracellular H2O2. - thioredoxin peroxidase, peroxiredoxin, hydrogen peroxide, TNF-alpha, NF-kappaB, apoptosis